The field of the present invention is the facile analysis of analytical samples. More particularly, the present invention relates to assessment of the quantity of a desired analyte using a catalyzed indicator reaction in competion with a second catalyzed reaction utilizing the same substrate as the indicator reaction.
The need for quantitative determination of the concentration of a substance of interest in a test sample is widespread and varied in application. Such need arises prominently in connection with clinical laboratory tests, quality control in the production of any mixture of materials, for example, food, agricultural, industrial, or other mixtures; and in analytical assessment of, for example, waste products such as industrial or municipal waste. Methods of quantitation are varied but generally involve generation of some detectable material such as a colored substance, a substance absorbing ultra-violet or infrared light at a particular wavelength, a fluorescent material, or, in general, a material which absorbs or generates radiation of a particular energy. Such radiation levels can be detected through instrumentation using instruments of complexity ranging from that of a simple colorimeter, or even a color comparator chart, to sophisticated variable wavelength specific infrared spectrometers. Additional detection methods rely on characteristic properties of either the substance to be determined or of a material into which it can be converted, which are assessed by more complex means such as, for example, mass spectroscopy, nuclear magnetic resonance spectroscopy, or stimulated emission of X-radiation.
The expense and sometimes the complexity of these analytical procedures are, of course, proportional to the sophistication of the instrumentation required, (although a highly complex instrument can sometimes eliminate many operator manipulations). Especially for preliminary screening tests, and for tests which are useful to individual consumers, it would be desirable to formulate testing procedures which obviate the need for expensive instrumentation and which are simple to perform. The present invention provides a method for quantitation of desired analytes which eliminates the need for such complication and expense. It provides a testing method which is useable unaided by instrumentally based analog measurements and gives essentially a digital readout useable to virtually anyone.
The method of the present invention is grounded in the competition for substrate by each of a pair of catalysts. Other procedures have recently been devised which depend on some sort of competitive reactivity, but which, nevertheless, often require quantitation using dedicated equipment. For example, the method of radioimmunoassay (RIA) capitalizes on competition between the desired analyte in a sample and supplied labeled analyte for the same antibody. By assessing the quantity of labeled material bound to antibody, the level of competition offered by the analyte in the sample can be assessed. This method requires that the operator use radioactive materials, and complex counting equipment is required. Similar procedures also based on competition for a specific immunoglobulin utilize a colorimetric detection system based on an enzymatic reaction. In such techniques, commonly known as ELISA or EMIT, competition from the analyte results in the release of enzyme an indicator reaction from an inactivating antibody.
In the present invention, however, the amount of analyte available for an indicator reaction is dependent on the amount of catalyst for a competing reaction present. By varying the amount of competing reaction catalyst the amount of analyte remaining can be left at a detectable level or not. As the amount of competing catalyst can be determined by the will of the experimenter (or by a series of preset levels chosen by the manufacturer), no instrumentation is required for its quantitation.